Use of live bacteria for growth promotion in animals

ABSTRACT

The present invention relates to the use of F4 +  non-pathogenic  Escherichia coli  strains to promote growth in an animal. The present invention also relates to the use of such strains to homogenize growth among a herd of animals. More specifically, the animal(s) of interest in the present invention are those wherein growth promotion or growth homogenization are desired goals, such as animals reared for meat production. The present invention further relates to a method for promoting growth of an animal as well as a method for homogenizing growth among a herd of animals.

FIELD OF THE INVENTION

The present invention relates to the field of growth promotion inanimals. More specifically, the present invention relates to the use ofa non-pathogenic Escherichia coli strain expressing the F4 (or K88)attachment factor, to either promote growth in animals or homogenizegrowth among a herd of animals.

BACKGROUND OF THE INVENTION

Growth promotion is a crucial issue for farm and breeding specialists,who mainly seek to optimize the production of healthy animals beforeslaughter or for research purposes. Such a concern should lead them touse growth promoting products that would prove beneficial to the animalsand also to humans, in the case of meat-producing animals.

One major caveat in farms and breeding environments is the weight loss,slow growth rate along with a recrudescence of concomitant diseases,drug cost, and mortality which lead to a decrease in animal yields andultimately to considerable economic losses. In this connection,post-weaning or post-hatching animals are particularly vulnerable toagents impeding growth.

Infections caused by either non-hygienic conditions or close proximitybetween animals, for example, are among the most common factors leadingto the above-mentioned caveat.

One conventional solution used to alleviate this problem has been to useantibiotic growth promoters in feeds. However, use of antibiotic growthpromoters is also highly controversial because, as is well known, evenat sub-therapeutic doses, continued antibiotic use can lead to selectionof antibiotic-resistant bacterial strains in the treated animals (ArnoldS et al.; Wegener HC et al. and Schwarz S et al.). In fact, in the lastten years, there has been an emergence of more pathogenic and moreantibiotic-resistant Escherichia coli strains and/or husbandry changes(early weaning) and/or new European regulations forbidding use ofantimicrobial agents as growth promoters or for prophylaxis treatment(prevention of diseases) and use of high levels of heavy metals, such aszinc oxide in the feed. The weaning period is particularly associatedwith higher antibiotic use during animal production.

Consequently, there is now a growing resistance to the use of antibioticgrowth promoters and heavy metals due to recrudescence of antibioticresistance, allergic reactions to antibiotic residues, and contaminationof cultivated soil.

Another caveat that farm and breeding specialists also have to face isgrowth heterogeneity among herds of animals. More specifically, in apurpose of optimized meat production for example, farmers seek toproduce consistent herds of animals displaying the most homogeneousgrowth rate possible before slaughter, to avoid increased costs.However, animals generally present different rates of growth anddifferent vulnerabilities to infectious agents, among others.

There is thus a constant need for innovating agents that promote growthof animals and that advantageously further contribute to homogenize andoptimize animal growth.

SUMMARY OF THE INVENTION

An object of the present invention is to use of an effective amount ofan F4+ non-pathogenic Escherichia coli strain to promote growth in ananimal.

Another object of the present invention is to use of an effective amountof an F4+ non-pathogenic Escherichia coli strain to homogenize growthamong a herd of animals.

A further object of the present invention is to provide a method ofpromoting growth in an animal, said method comprising the step offeeding said animal with an effective amount of an F4+ non-pathogenicEscherichia coli strain.

Yet another object of the present invention is to provide a method ofhomogenizing growth among a herd of animals, said method comprising thestep of feeding said animals with an effective amount of an F4+non-pathogenic Escherichia coli strain.

Owing to the use of F4+ non-pathogenic Escherichia coli strains, theinvention finds an advantage in situations wherein rapid growthpromotion and growth homogenization of an animal are particularlyneeded. For instance, use of F4⁺ non-pathogenic Escherichia coli strainsin animals reared for meat production allows to bring these animals tomarket weight or slaughter weight in a shorter growing period than thatof their untreated counterparts.

The present invention may further find an advantage for growth promotionand growth homogenization of laboratory animals, such as rats and mice.As can be appreciated, bringing these laboratory animals to a givenweight faster and more homogeneously preferably provides for morehomogeneous and readily available samples of animals.

Another advantage of the present invention is that there is no recourseto antibiotic use to promote growth in animals. Therefore, problems suchas development of antibiotic-resistant bacterial strains or allergies toantibiotics which particularly affect post-weaning animals, arealleviated.

Moreover, since heavy metals such as zinc oxide are not added to theanimal's feed, contamination of the soil is also avoided. In otherwords, the present invention also provides for environment-friendly usesand methods.

The improved efficiency of feed conversion attained by the presentmethod enables treated animals to reach any desired weight whileconsuming less food than untreated animals grown to the same weight.Moreover, while practicing the method of the present invention, neithertoxic side effects nor decrease in general health status due to thebacterium is observed in the treated animals.

The invention and its advantages will be better understood upon readingthe following non-restrictive description of preferred embodimentsthereof, made with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the pulsed-field gel electrophoresispattern of Xbal-digested genomic DNA of a preferred F4⁺ non-pathogenicEscherichia coli strain used in accordance with the present invention.The preferred strain is the coliPROtec strain.

FIG. 2 is a graph that displays the percentage of weight gain in pigsfor each period after treatment with F4⁺ Escherichia coli strains.

FIG. 3 is a graph that displays the percentage of weight gain in miceafter treatment with F4⁺ Escherichia coli strains.

DETAILED DESCRIPTION OF THE INVENTION

The originality of the present invention stems from new uses for F4⁺non-pathogenic Escherichia coli strains. More particularly and accordingto a first aspect, the present invention relates to the use of aneffective amount of an F4⁺ non-pathogenic Escherichia coli strain topromote growth in an animal.

According to a second aspect, the present invention relates to the useof an effective amount of an F4⁺ non-pathogenic Escherichia coli strainto homogenize growth among a herd of animals.

According to related aspects, the present invention relates to a methodof promoting growth in an animal and to a method of homogenizing growthamong a herd of animals.

Both methods comprise the step of feeding the animal(s) with aneffective amount of an F4⁺ non-pathogenic Escherichia coli strain.

The strain used in the present invention is characterized in that itexpresses the attachment factor F4, while being non-pathogenic.According to a preferred aspect of the invention, the F4⁺ non-pathogenicEscherichia coli strain is selected from the group consisting ofcoliPROtec (Accession number IDAC 210105-01), JG1329, M226,P03-7586(175) or pMK005. More preferably, the present inventioncontemplates using the coliPROtec strain and/or mutants or variantsthereof (see Example I for more details).

As used herein, the terms “mutants” and “variants of the coliPROtecstrain are used for strains that have all the identifyingcharacteristics of the coliPROtec strain, as provided in Example I.Mutant or variant strains may be identified as having a genome or partthereof that hybridizes under conditions of high stringency to thegenome of the coliPROtec strain.

According to the above-mentioned aspects of the invention, theabove-described strain is used in an “effective amount”. By theexpression “effective amount”, it will be understood that the amount ofan F4⁺ non-pathogenic Escherichia coli strain is the amount that willelicit the biological response of a tissue, system or animal that isbeing sought by the researcher or the veterinarian, for example. Inother words, such an effective amount of an F4⁺ non-pathogenicEscherichia coli strain is the amount that is sufficient for promotinggrowth in an animal as well as homogenizing growth among a herd ofanimals.

It will be understood that a preferred effective amount of the straincontemplated by the present invention is at least about 5^(E)7colony-forming units (CFU), and more preferably range from about 5^(E)7to about 5^(E)9 CFU of the strain of interest per animal. By “about”, itis meant that the CFU value of said strain can vary within a certainrange depending on the margin of error of the method used to evaluatethe number of CFU for such a strain.

The effective amount to be used may vary according to a number offactors. For instance, type of animal, initial weight of the animal,growth phase of the animal, environment i.e. animal facilities, type andmanagement of production, hygienic status of the facilities, stressafter weaning or hatching, feed and supplements used, health of theanimal and concomitant diseases or treatment, may be factors to takeinto account.

As mentioned above, the objectives of growth promotion and growthhomogenization are achieved in an animal. As used herein, the term“animal” refers to any young or adult animal suitable to be used inaccordance with the present invention. More preferably, the term“animal” refers to a post-weaning or a post-hatching animal.

Since in either a post-weaning or post-hatching animal, maternal feedingand caring are no longer available, growth promoting agents are of theutmost importance. Such a concern is particularly crucial, for instance,in the case of animals bred for their meat.

In the context of the present invention, preferred animals to be usedcan be any one of the following: farm animals such as pigs and morepreferably post-weaning pigs; poultry such as chickens, ducks, geese,hens or turkeys, preferably, chickens and more preferably broilerchickens; cattle such as cows, steers, bulls or oxen; game animals suchas ostriches or bisons; domestic animals, for example cats or dogs; andlaboratory animals, such as mice or rats. Of course, these animals areall characterized in that growth promotion and growth homogenization aredesired features, in particular when meat production is the goal.

In view of the above, it will be understood that the animals may receivethe strain of the invention for substantially the whole of their growingperiod, or for only a part of their growing period, for example theearly part and/or the period leading up to slaughter.

According to a preferred aspect of the invention, the post-weaninganimal is preferably a pig, preferably aged from about 10 to about 28days old, at the onset of the trials. The pig of interest is morepreferably 17 days old.

According to another preferred aspect of the invention, the post-weaninganimal is preferably a mouse, preferably aged from about 18 to about 28days old, at the onset of the trials. The mouse of interest is morepreferably 21 days old.

According to yet another preferred aspect of the invention, thepost-hatching animal is preferably a chicken, preferably aged from about1 to about 7 days old, at the onset of the trials. The chicken ofinterest is more preferably 1 day old. It will be understood that theexpression “about 1 day old” means 24 hours or less after birth orhatching.

According to a preferred aspect of the present invention, the effectiveamount that can be given to pigs preferably varies from 5^(E)7 to 5^(E)9CFU/pig and is more preferably about 1^(E)9 CFU/pig.

According to another preferred aspect, the effective amount that can begiven to chickens varies from about 5^(E)7 to 5^(E)9 CFU/chicken, and ismore preferably 5^(E)8 CFU/chicken.

According to yet another preferred aspect, the effective amount that canbe given to mice varies from about 5^(E)7 to 5^(E)9 CFU/mouse, and ismore preferably 5^(E)8 CFU/mouse.

In the particular context of the methods of the invention, the effectiveamount of the strain may be fed to the animal as a single dosage or maybe given according to a regimen, whereby it is effective. By the term“feeding”, it should be understood that an F4⁺ non-pathogenicEscherichia coli strain of the invention is provided to the animal undertreatment so that the strain eventually reaches the gastrointestinaltract, and more preferably the intestines.

For instance, and according to a preferred aspect of the invention,feeding can be done by orally feeding the strain to the animal ofinterest.

According to a preferred aspect of the invention, the strain ispreferably fed to the animal in lyophilized form. According to anotherpreferred aspect, the strain of the invention is diluted or suspendedwith a diluent or carrier.

In accordance with the present invention, the F4⁺ non-pathogenicEscherichia coli strain is used to “promote growth” in an animal or to“homogenize growth among a herd animals”. These expressions refer to theuse of the Escherichia coli strain to increase an animal's growth rate.In other words, upon treatment with the strain of interest, the animal'sweight increases more rapidly and more homogeneously compared with itsuntreated counterparts. More particularly, the expression “growthhomogenization” in the context of the present invention refers to arelative control over the rate of growth in a herd of animals. This typeof control bears a particular interest in the field of animal breedingand meat production in which all the animals in a given group mustideally reach their growth peak faster, at relatively the same timeamong the group, with conventional amounts of feed along with anon-toxic growth promoting agent. When these conditions are met, feedingcosts, slaughter costs and shipping costs can all be optimized.

In order to evaluate growth promotion and growth homogenization, anumber of parameters known in the field can be determined. Morespecifically, such parameters, evaluated either alone or in combination,can encompass:

-   -   i—Mean body weight (MBW): mean of the body weights of a group of        animals;    -   ii—Mean daily weight gain (DWG): mean increase in weight per        day, per group of animals, during a particular period;    -   iii—Feed intake (FI): quantity of feed ingested per animal or        per group of animals during a particular period; and    -   iv—Feed conversion ratio (FCR): feed intake per animal or per        group of animals for a particular period/Weight gain for the        same animal or group of animals for the same particular period.

Advantageously, the strain of the invention may be used alone or inassociation with a feed acceptable carrier. As used herein, theexpression “feed acceptable carrier” refers to any carrier, diluent orexcipient that is compatible with the strain of the invention and can begiven to an animal without adverse effects. Suitable feed acceptablecarriers known in the art include, but are not limited to, water,saline, glucose, dextrose, or buffered solutions. Such a carrier isadvantageously non-toxic to the strain and not harmful to the animal. Itmay also be biodegradable. A person skilled in the art will know how toselect suitable carriers, such as carriers that are not harmful to theenvironment. Preferably also, this carrier is a suitable solid or liquidfeed acceptable carrier.

A suitable solid feed acceptable carrier is a non-toxic ingestablecarrier. For instance, this solid feed acceptable carrier may be acommon solid feedstuff such as the component of a typical animal dietconsisting of cereal products, such as barley meal, maize meal or wheatfeed, nut and seed products, such as decorticated ground nut cake orcotton seed cake, or extracted cotton seed cake, together with minoramounts of, for example, feather meal, seaweed meal, bone meal, boneflour, chalk, salt, urea and vitamins; or it may be an inert soliddiluent or carrier of no nutritional value, for example kaolin, talc,calcium carbonate, fuller's earth, attapulgus clay, ground oyster shellsor ground limestone; or it may be starch or lactose.

A suitable liquid feed acceptable carrier is, for example, water andpreferably drinking water; milk such as whole or skim milk; or a culturemedium such as a trypsone soy broth (TSB).

The following examples illustrate the wide range of potentialapplications of the present invention and are not intended to limit itsscope. Modifications and variations can be made therein withoutdeparting from the spirit and scope of the invention. Although anymethod and material similar or equivalent to those described herein canbe used in the practice for testing the present invention, the preferredmethods and materials are described.

EXAMPLES Example I Description of the Escherichia coli strain(coliPROtec strain) and its clones

According to a preferred feature of the invention, an F4⁺ non-pathogenicEscherichia coli strain (referred hereinafter as the coliPROtec or theJFF4 strain) is used. The coliPROtec strain was deposited at theInternational Depositary Authority of Canada on Jan. 21, 2005 and wasattributed accession number IDAC 210105-01. The address for theInternational Depositary Authority of Canada is 1015 Arlington Street,Winnipeg, Canada, R3E 3R2.

1. Origin

The strain was isolated from feces of a healthy pig at the Escherichiacoli Laboratory at the Faculty of veterinary medicine, University ofMontreal, Saint-Hyacinthe, Quebec, Canada. Animals were purchased from alocal farm located in Monteregie, Quebec, Canada.

2. Stability of the Strain

The expression of F4 fimbriae, an immunogenic protein, of the coliPROtecstrain was stabilized using in vitro passages. Three (3) consecutive 10L fermentations were done. After each fermentation, the culture wasinoculated on agar and 10 colonies were tested for F4 fimbriaeexpression using a slide agglutination test. Bacteria from F4⁺ colonieswere pooled and used for the consecutive fermentation. The strain wasthen frozen in trypsone soy broth (TSB) supplemented with 20% glycerol.After thawing, the F4 expression of the strain was not totally stableand several consecutive fermentations (approximately 10) were needed toobtain a stable strain. The stable strain was freeze-dried afterapproximately 15 passages from the pig. This freeze-dried culture wasused to produce the Master Seed of coliPROTec.

3. Biochemical Analysis

An identification of the strain was done using the API system. Theidentification code 5544572 obtained referred as an Escherichia colistrain.

4. Virotyping

Virotyping of the coliPROtec strain was done by colony hybridizationand/or polymerase chain reaction (PCR). Virotyping results showed thatthe coliPROtec strain was positive for F4 whereas it was negative forthe following toxins:

-   LT, STa, STb, STaH, Stx1, Stx2, VT2vp1, VT2vh, Aero, Tsh, CDT3,    CDT4, CNF1, CNF2, HlyA, HlyC, Ehx, East1.

Furthermore, the coliPROtec strain was also negative for the followingadhesins or putative adhesins:

-   F5, F6, F18, F41, F17, P fimbriae, AIDA, AFA, SFA, CS31a, daaE, Paa,    aggR, ARF/1, Eae, CFAI, CFAII(CS1coo), CFAII(CS3cst).    5. DNA Fingerprinting

The coliPROtec strain was characterized using pulse-field gelelectrophoresis (FIG. 1).

Particular clones of the strain, for which the F4 fimbriae expressionwas stable after fermentation, were selected following repeated in vitropassages.

6. Summary Table and Data Sheet

The strains used in the following experiments are described in detail inthe following Table 1 and Data Sheet.

TABLE 1 Name Serotype Source F4⁺ non pathogenic wild type Escherichiacoli strains JFF4 (coliPROtec) O8:K87 feces of a healthy pig JG1329O8:K87 feces of a pig M226 O9 feces of a pig P03-7586(175) O8 feces of apig F4⁺ non pathogenic recombinant Escherichia coli strain pMK005 O9plasmid pMK005 coding for F4 F4-negative non pathogenic Escherichia colistrain P82-862 O115:KV165 feces of pig DATA SHEET of the coliPROtecstrain Determined with standard method of serotyping using “O” antigenand at the “Statens Serum Institut 5 Artillerivej 2300 Copenhagen SDenmark” PATHOTYPE NEGATIVE FOR THE FOLLOWING TOXINS LT, STa, STb, STaH,Stx1, Stx2, VT2vp1, VT2vh, Aero, Tsh, CDT3, CDT4, CNF1, CNF2, HlyA,HlyC, Ehx, East1 NEGATIVE FOR THE FOLLOWING VIRULENCE FACTORS F5, F6,F18, F41, F17, P fimbriae, AIDA, AFA, SFA, CS31a, daaE, Paa, aggR,ARF/1, Eae, CFAI, CFAII(CS1coo), CFAII(CS3cst) Confirmation by PCR andcolony hybridization MEDIUM FOR FIMBRIAE EXPRESSION ANTIBIOGRAMAmpicillin, tetracycline, spectinomycin, tiamulin, tylosine RESISTANTTO: ANTIBIOGRAM Apramycin, ceftiofur, cephalothin, gentamicin, neomycin,SENSITIVE TO: trim/sulfamethoxazol STORAGE OF MEDIUM Freeze dried(lyophilization) medium: 5% dextran T-40, 7% saccharose, 1% monosodiumglutamate SOURCE OF THE Isolated at The Escherichia coli Laboratory,Fac. méd. vét., Saint- ISOLATE Hyacinthe, 1999, from feces of a normalpig.

Example II Weaned Pigs Effects of F4⁺ Non-Pathogenic Escherichia coliStrains, in Oral Form, on the Growth of Pigs

A—Effect of coliPROtec on Weight Gain in Weaned Pigs

1. Escherichia coli Strain:

The live Escherichia coli F4⁺ non-pathogenic strain coliPROtec suspendedin TSB was orally fed to weaned pigs. This strain is described inExample I.

2. Experiments

2.1 Animals

Five (5) trials were performed for a total of 45 treated and 45untreated pigs. These pigs came from different commercial farms.

2.2 Trials

The trials were conducted at the Faculté de médicine vétérinaire,Université de Montréal, under the following schedule.

2.2.1 Trial Groups

GROUP NO. (n) DESCRIPTION 1 (45) Untreated 2 (45) Treated withcoliProtec (with 5^(E)9/pig)2.2.2 Trial Schedule

DAY NO. DESCRIPTION 1 Arrival of the 17-day-old weaned pigs. 1 To 4Adaptation period 5 Treatment of the animals with a single dose ofcoliPROtec or TSB only (control group) 20  Weighing of the animals; Endof the experiment.2.2.3 Evaluated Parameters

In the present assays, the parameters evaluated were the weight and thedaily weight gain of the weaned pigs on days 5 and 20 in Groups 1 and 2.

3. Results

During the innocuity studies of the coliPROtec strain, a positive effectof the product was observed on the weight gain of the animals. As shownin Table 2 and 3, pigs treated with only one oral dose of coliPROtec atthe beginning of the post-weaning period had a daily weight gain higherby 53 g when compared to untreated animals. Specifically, 2 weeks afterthe treatment, treated pigs were 849 heavier than untreated pigs.

TABLE 2 Weight of animals Weight (Kg) Group 2 vs Day Group 1 Group 2T-test Group 1 (g) 5 6.053 6.137 p = 0.754 +84 20 12.055 12.904 p =0.020 +849

TABLE 3 Daily weight gain Daily weight gain (g) Group 2 vs Days Group 1Group 2 T-test Group 1 (g) 5 to 20 405 458 p = 0.007 +534. Analysis and Conclusion

At day 5 post-weaning, giving coliPROtec, the weight of the Groups 1 and2 (untreated and treated with coliPROtec) was not statisticallydifferent. However, at day 20 post-weaning (15 days after thetreatment), the treated animals were 849 g heavier and demonstrated adaily weight gain of 53 g more than untreated animals. These differenceswere statistically significant.

Of note, the conventional antibiotic growth promoters generally increasethe weight gain by 3.3 to 8.8% (Doyle, M. E., Food Research Institute,University of Wisconsin, 2001). As demonstrated here, the coliPROtecstrain increased the daily weight gain by 11% during the 2 weeksfollowing the single dose.

B—Effects of Live F4⁺ Non Pathogenic Escherichia coli Strains, in OralForm, on the Growth of Weaned Pigs

1. Escherichia coli Strains

The strains used are described in Example I.

2. Experiments

2.1. Animals

Forty nine (49) 17-day-old weaned pigs, originating from a clean,conventional pig farm, were used in the present experiments. Thesepiglets had a body weight of 5±1 kg.

2.2. Trials

2.2.1 Trial Groups

At weaning, the pigs were transferred into the animal facilities,containment rooms, Laboratoire d' Hygiène Vétérinaire et Alimentaire,Saint-Hyacinthe, Quebec, Canada. Laboratory analyses were led at the EcLLaboratory, FMV, Saint-Hyacinthe, Quebec, Canada.

GROUP no. (n) DESCRIPTION 1 (7) untreated pigs 2 (7) treated with theF4-negative non pathogenic Escherichia coli strain P82-862 3 (7) treatedwith the F4⁺ non pathogenic Escherichia coli JFF4 strain (coliPROtec) 4(7) treated with the F4⁺ non pathogenic Escherichia coli JG1329 strain 5(7) treated with the F4⁺ non pathogenic Escherichia coli M226 strain 6(7) treated with the F4⁺ non pathogenic Escherichia coli P03-7586(175)strain 7 (7) treated with the F4⁺ non pathogenic recombinant Escherichiacoli pMK005 strain.2.2.2 Trial Schedule

DAY no. DESCRIPTION 0 Arrival of the 17-day-old weaned pigs at theanimal facilities. 1 Identification, grouping and weighing of pigs.Grouping according to animal sex and weight. Sampling of feces forevaluation of the excretion of F4, LT, STa and/or STb positiveEscherichia coli (PCR) 1 and 4 Approximately 5E9 CFU of Escherichia colibacteria in trypsone soy broth (TSB) per pig given orally using anoesophageal tube. The control group received TSB only. 1, 4, 9, 14Weighing of the animals; sampling of feces for evaluation and 17 of theexcretion of F4, LT, STa and/or STb positive Escherichia coli (PCR) 17 Euthanasia of animals2.2.3 Evaluated Parameters

During the trial, pigs had ad libitum access to feed and water. Theywere observed twice daily for general health and presence of diarrhea.From day 0 to day 14, pigs were fed with a commercial starter feedcontaining 23% protein without addition of zinc oxide or antibiotics.For the last 3 days, the feed contained 19% protein.

3. Results

3.1 F4⁺ Escherichia coli and Pathogenic ETEC Status of Pigs

At the beginning of the trial, thus before treatment, some pigs from allgroups were colonized by an F4⁺ strain possessing the toxin STb (Table4). All pigs of the control group (Group 1) and of the group treatedwith an F4⁻ Escherichia coli strain (Group 2), were colonized by thisF4:STb strain at day 9 (Table 5). Although this strain (O45:F4:STb) isnot a usual ETEC strain causing post-weaning diarrhea in pigs, we cannot exclude that it is pathogenic. However, no animal demonstratedclinical signs associated with post-weaning diarrhea during the trial.

Since both control groups were colonized by this F4⁺ strain, it isdifficult to evaluate the effect of the tested F4⁺ strains on animalgrowth performance.

TABLE 4 Identification of the status of pigs for excretion of F4⁺Escherichia coli strain before treatment (Day 1; PCR analysis on feces)Number of pigs with Other virulence factors feces positive for F4identified Group 1*¹ 1 LT and STb Group 2 1 STb Group 3 2 LT and STbGroup 4 1 LT and STb Group 5 1 LT and STb Group 6 3 LT and STb Group 7 1STb *¹Treatment: Group 1; control, Group 2; F4-negative Escherichia Colistrain at days 1 and 4, Groups 3 to 7; F4⁺ strains at days 1 and 4.

TABLE 5 Identification of the status of pigs for excretion of F4⁺Escherichia coli strains during the trial Number of pigs with fecespositive for F4 Day 1*¹ Day 4 Day 9 Day 14 Day 17 Group 1*¹ 1 3 7 4 0Group 2 1 2 7 3 0 Group 3 2 4 6 5 5 Group 4 1 7 7 6 0 Group 5 1 6 6 5 6Group 6 3 1 1 7 4 Group 7 1 1 1 0 0 *¹Treatment: Group 1; control, Group2; F4-negative Escherichia coli strain at days 1 and 4, Groups 3 to 7;F4-positive strains at days 1 and 4.3.2 General Health and Diarrhea Assessment

All animals were in good health during the trial. Some animals of groups2, 3, 4, 6 and 7 presented mild diarrhea, but for only one day.

3.3 Growth Performance Assessment

TABLE 6 Weight of pigs after treatment with F4⁺ Escherichia coli strainsBody weight (Kg) Day 1*1 Day 4 Day 9 Day 14 Day 17 Group 1*¹ 4.91 5.436.74 9.09 10.23 Group 2 4.86 5.40 7.11 9.23 11.20 Group 3 4.91 5.77 7.239.34 10.91 Group 4 5.00 5.13 7.07 9.20 10.60 Group 5 4.97 5.59 7.04 9.2710.47 Group 6 4.91 5.47 7.20 9.41 10.46 Group 7 5.03 5.80 8.03 10.2011.23 *¹Treatment: Group 1; control, Group 2; F4-negative Escherichiacoli strain at days 1 and 4, Groups 3 to 7; F4-positive strains at days1 and 4.

The linear model with repeated measures, using the day as within-subjectfactor and the group as between-subject factor, showed no effect oftreatment on growth of pigs (p=0.90). Post hoc analysis checked fordifferences between each treated group (groups 2 to 7) and the controlgroup (group 1) at each day (Table 6). Weight was significantly higherfor group 7, compared to the control group, at day 9 only (p=0.045).Other weight differences were not significant.

Nevertheless, Groups 3 and 7, treated with the JFF4 and the recombinantstrains, respectively, had a higher weight at days 4 and 9, but weightswere subsequently more similar between groups (Table 6). During thefirst period of the trial (days 0 to 4), groups 3 and 7 had a percentageof weight gain of 18 and 15%, respectively, compared to 11% for bothcontrol groups (Groups 1 and 2; FIG. 2). The low weight gain observedfor the group 4 during this first period was attributed to 3 pigs thatlost weight.

During the second period (days 4 to 9), groups 2, 4, 6 and 7 had ahigher percentage of weight gain than that of the untreated group (group1). The percentage of weight gain was more homogeneous during the thirdperiod (days 9 to 14) and was higher for groups 2 and 3 than for group1, during the last period.

Example III Broiler Chickens Effects of Live F4⁺ Non PathogenicEscherichia coli Strains, in Oral Form, on the Growth of BroilerChickens

1. Escherichia coli Strains

The strains used are described in Example I.

2. Experiments

2.1 Animals

Sixty-three (63) male 1-day-old Cobbs broiler chicks, originating from aclean, conventional chicken farm. After hatching, chicks weretransferred into animal facilities.

2.2 Trials

2.2.1 Trial Groups

At hatching, the chickens were transferred into the animal facilities,containment rooms, Faculté de médecine vétérinaire, Saint-Hyacinthe,Quebec, Canada.

Laboratory analyses were led at the EcL Laboratory, FMV,Saint-Hyacinthe, Quebec, Canada.

GROUP NO. (N) DESCRIPTION 1 (10) untreated chickens 2 (10) treated withthe F4-negative non pathogenic Escherichia coli strain P82-862 3 (10)treated with F4⁺ non pathogenic Escherichia coli JFF4 strain(coliPROtec) 4 (10) treated with the F4⁺ non pathogenic Escherichia coliJG1329 strain 5 (10) treated with the F4⁺ non pathogenic Escherichiacoli M226 strain 6 (10) treated with the F4⁺ non pathogenic Escherichiacoli P03-7586(175) strain 7 (10) treated with the F4⁺ non pathogenicrecombinant Escherichia coli pMK005 strain.2.2.2 Trial Schedule

DAY NO. DESCRIPTION 0 Arrival of the 1 day-old chicks at the animalfacilities; identification, Weighing and grouping of chicks; Sampling offeces for evaluation of the excretion of F4, LT, STa and/or STb positiveEscherichia coli (PCR) 1 and 4 Approximately 1E9 CFU of Escherichia colibacteria in trypsone broth (TSB) per chick given orally using anoesophageal needle. The control group received TSB only 0, 4, 9,Weighing of the animals; evaluation of the excretion of F4, 14, 18, 23LT, STa and/or STb positive Escherichia coli (PCR) and 28 2, 9, 17Evaluation of the 24 hour feed consumption and 24 28  Euthanasia ofanimals2.2.3 Evaluated Parameters

Mean body weight (MBW), Mean daily weight gain (DWG), feed intake (FI),and feed conversion ratio (FCR) were all evaluated in the presentassays.

During the trial, chickens had ad libitum access to feed and water. Theywere observed twice daily for the general health and presence ofdiarrhea. From day 0 to day 24, chickens received a standard commercialfeed for chicks (without antibiotics). From day 24 to day 28, theyreceived a standard development commercial feed (without antibiotics).

3. Results

3.1 F4⁺ Escherichia coli and Pathogenic ETEC Status of Chickens

No fecal sample was positive for F4, STa, STb or LT at day 0, beforetreatment. Fecal excretion of F4⁺ Escherichia coli was detected intreated groups (groups 3 to 7) at days 2 and 4, but not subsequently.

3.2 General Health and Diarrhea Assessment

Animals were in good health during the trial and no diarrhea wasobserved. Three (3) animals of group 7 were euthanized at day 23 or 25due to their deteriorating general health status. No gastro-intestinalclinical sign was observed in these chickens. Necropsy reports on thesechickens from the Pathology department (Faculté de médecine vétérinaire,Saint-Hyacinthe, Quebec, Canada) revealed that these chickens died fromthe ascites syndrome, a frequent non infectious disease, generallyassociated with cardiac insufficiency, in broiler chickens.

3.3 Growth Performance Assessment

TABLE 7 Weight of chickens after treatment with F4⁺ Escherichia colistrains

^(*1)Treatment: Group 1; control, Group 2; F4-negative Escherichia colistrain at days 1 and 4, Groups 3 to 7; F4-positive strains at days 1 and4. ^(*2)Three euthanized animals were excluded

TABLE 8 Daily weight gain of chickens after treatment with F4⁺Escherichia coli strains

^(*1)Treatment: Group 1; control, Group 2; F4-negative Escherichia colistrain at days 1 and 4, Groups 3 to 7; F4-positive strains at days 1 and4. ^(*2)Three euthanized animals were excluded

Strain JFF4: The MBW of group 3, treated with the strain JFF4, washigher than for the untreated group (group 1) and the group treated withthe F4-negative strain (group 2) on days 9, 14, 18 (Table 7). The DWG ofthe group treated with strain JFF4 was higher than for both controlgroups (groups 1 and 2) during the days following the second treatment(period between days 4 and 9) and during the fourth period (days 14 to18; Table 8).

Other F4³⁰ strains: The MBW of groups 5, 6, and 7 was higher than thatof both control groups (groups 1 and 2) on days 6, 14, 18, and 23. Bycontrast, it was higher only on day 9 for group 4 (Table 7).

The DWG of all groups treated with F4⁺ strains was higher than that ofboth control groups during the days following the treatments (days 4 to9) and during the fourth period (days 14 to 18) for group 5 (Table 8).

F4-negative Strain:

The MBW and DWG were similar or lower for the group treated with theF4-negative strain than for the untreated group.

A linear model with repeated measures, using the day as within-subjectfactor and the group as between-subject factor, showed no effect oftreatment on growth of chickens (p=0.97). Post hoc analysis was done tocheck for differences between each treated group (groups 2 to 7) and theuntreated group (group 1) on each day. Weight was not significantlydifferent between each treated group and the untreated group on eachday. Differences observed in the descriptive analysis were notsignificant probably due to a higher variability than expected for theweight of chickens in each group, in particular for the untreated group.

3.4 Feed Intake (FI)

TABLE 9 Feed intake (24-hr-period) of chickens after treatment with F4⁺Escherichia coli strains

^(*1)Treatment: Group 1; control, Group 2; F4-negative Escherichia coilstrain at days 1 and 4, Groups 3 to 7; F4-positive strains at days 1 and4. ^(*2)Two euthanized animals (n = 7 instead of 9)

Strain JFF4: The FI of group 3 (JFF4) was lower than that of both theuntreated group (group 1) and the group treated with the F4-negativestrain (group 2) at days 2, and 17 (Table 9).

Other F4⁺ strains: The FI was lower than that of both control groups(groups 1 and 2) only for group 4 (days 2, 9, 17) and for group 6 (day17).

F4-negative strain: The FI of the group treated with an F4-negativestrain was lower than that of the untreated group (group 1) at days 9,17, and 24.

A linear model showed no effect of treatment on feed consumption ofchickens (p=0.77).

3.5 Feed Conversion Ratio (FCR)

TABLE 10 Feed conversion ratio of chickens after treatment with F4⁺Escherichia coli strains

^(*1)Day 2; total DWG of the group between days 0 and 4/FI at day 2, Day9; total DWG of the group between days 4 and 14/FI at day 9, Day 17;total DWG of the group between days 14 and 23/FI at day 17, Day 24;total DWG of the group between days18 and 28/FI at day 24.^(*2)Treatment: Group 1; control, Group 2; F4-negative Escherichia coilstrain at days 1 and 4, Groups 3 to 7; F4-positive strains at days 1 and4. ^(*3)Two euthanized animals (n = instead of 9)

Strain JFF4: The FCR of group 3 (JFF4), was lower than that of both theuntreated group (group 1) and the group treated with the F4-negativestrain (group 2) at days 2, and 17 (Table 10), similarly to the FI(Table 9). This ratio was lower than that of the untreated group, onlyat day 9.

Other F4⁺ strains: The FCR was lower than that of both control groups(groups 1 and 2) for group 7 (day 2), groups 3 and 4 (days 9 and 17),and groups 3 and 6 (day 17; table 4)

F4-negative strain: The FCR of the group treated with an F4-negativestrain was lower than that of the untreated group (group 1), at day 17only.

A linear model showed no effect of treatment on the feed conversionratio of chickens (p=0.68).

4. Analysis and Conclusion

Results demonstrate that F4⁺ non-pathogenic E. coli strains, includingthe JFF4 strain, increase the growth performance of chickens. The growthperformance was positively affected, especially for the days immediatelyfollowing the second treatment, the DWG being higher for all treatedgroups than for the untreated group, during days 4 to 9. Two F4⁺strains, including the JFF4 strain, also had a higher DWG than bothcontrol groups during days 14 to 18. The improvement in the growthperformance during the days immediately following treatments (days 4 to9) resulted in a higher weight for groups treated with F4⁺ strains thanfor both control groups until day 18 or, for some groups, day 23.

The higher DWG observed during the short period post-treatment affectedpositively the MBW of treated groups until days 18 or 23, depending onthe strain. Greater weight of treated groups was not associated withhigher feed intake. Furthermore, FI was sometimes lower for the treatedgroups than for the untreated group, thus lowering the feed conversionratio.

This effect on growth performance is associated with the F4 determinantor with strains expressing the F4 determinant since the group treatedwith the F4-negative Escherichia coli strain did not show this effectand had similar growth performance to the untreated group.

Example IV Weaned Mice Effects of Live F4⁺ Non Pathogenic Escherichiacoli Strains, in Oral Form, on the Growth of Weaned Mice

1. Escherichia coli Strains

The strains used are described in Example I.

2. Experiments

2.1 Animals

Seventy (70) healthy 21-day-old weaned mice. At weaning, mice weretransferred into the animal facilities.

2.2 Trials

2.2.1 Trial Groups

At weaning, the mice were transferred into the animal facilities,containment rooms, Laboratoire d'Hygiène Vétérinaire et Alimentaire,Saint-Hyacinthe, Quebec, Canada. The trials were also led at the EcLLaboratory, FMV, Saint-Hyacinthe, Quebec, Canada.

GROUP NO. (N) DESCRIPTION 1 (10) untreated mice 2 (10) treated with theF4-negative non pathogenic Escherichia coli strain P82-862 3 (10)treated with the F4⁺ non pathogenic Escherichia coli JFF4 strain(coliPROtec) 4 (10) treated with the F4⁺ non pathogenic Escherichia coliJG1329 strain 5 (10) treated with the F4⁺ non pathogenic Escherichiacoli M226 strain 6 (10) treated with the F4⁺ non pathogenic Escherichiacoli P03-7586(175) strain 7 (10) treated with the F4⁺ non pathogenicrecombinant Escherichia coli pMK005 strain.2.2.2 Trial Schedule

DAY NO. DESCRIPTION 0 Arrival of the 21 day-old weaned mice at theanimal facilities; identification, weighing and grouping of mice; foreach treated group, 5 males and 5 females were grouped in 2 cages.Sampling of feces for evaluation of the excretion of F4, LT, STa and/orSTb positive Escherichia coli (PCR) 1and 4 Approximately 1E9 CFU ofEscherichia coli bacteria in trypsone broth (TSB) per mice given orallyusing an oesophangeal needle. The control group received TSB only. 1, 4,9, Weighing of the animals; evaluation of the feed consumption 14, 18,23 for the period from the day of the previous weighing to and 28 theday of the weighing. Feces were sampled for evaluation of excretion ofF4, LT, STa and/or STb positive Escherichia coli (PCR) 28 Euthanasia ofanimals

Variables evaluated: Mean body weight gain (MBW), feed intake (FI), andfeed conversion ratio (FCR).

During the trial, mice had ad libitum access to feed and water. Theywere observed twice daily for general health and presence of diarrhea.

3. Results

3.1. F4⁺ Escherichia coli and Pathogenic ETEC Status of Mice

No fecal sample was positive for F4, STa, STb or LT at day 0. F4 wasidentified in the feces of groups 3, 4, 5 and 7 on the day after thefirst and/or up to 10 days after the second treatment with the F4⁺strains. No STa, STb or LT was detected at any time during theexperiment.

3.2. General Health and Diarrhea Assessment

All animals were in good health during the trial and no diarrhea wasobserved.

3.3. Growth Performance Assessment

TABLE 11 Mean body weight of mice after treatment with F4⁺ Escherichiacoli strains Body weight (g) Day Group 0 Day 4 Day 9 Day 14 Day 18 Day23 Day 28 ^(‘) 1*¹ 16.71 21.01 23.67 24.95 26.15 27.35 28.24 2 17.2021.67 24.40 25.72 26.81 28.32 29.38 3 17.33 21.93 24.42 25.85 27.1027.88 28.94 4 17.54 21.39 24.37 26.12 26.87 28.09 29.48 5 18.05 21.6824.40 25.70 27.42 28.85 29.90 6 17.77 21.52 24.37 25.83 26.92 27.8929.13 7 17.79 20.88 23.44 24.03 26.15 27.41 28.29 *¹Treatment: Group 1;control.Group 2; F4-negative Escherichia coli strain at days 1 and 4,Groups 3 to 7; F4-positive strains at days 1 and 4.

No difference in body weight was observed between groups. Linear modelwith repeated measures, using the day as within-subject factor and thegroup as between-subject factor, showed no effect of treatment on growthof mice (p=0.99). Post hoc analysis checked for differences between eachtreated group (groups 2 to 7) and the control group (group 1) on eachday. Weight was not different between each treated group and the controlgroup on each day.

Percentages of weight gain of all groups were similar during the trialperiod of 28 days. On the other hand, groups 1, 2, and 3 had slightlygreater increase, at day 4 only (FIG. 3).

3.4 Feed Intake (FI)

TABLE 12 Feed intake of mice after treatment with F4⁺ Escherichia colistrains

^(*1)Treatment: Group 1; control, Group 2; F4-negative Escherichia colistrain on days 1 and 4, Groups 3 to 7; F4⁺ strains on days 1 and 4.

Strain JFF4: The FI of group 3 (JFF4), was lower than that of both theuntreated group (group 1) and the group treated with the F4-negativestrain (group 2) between days 4 to 9, early after treatment. However, itwas lower than that of the untreated group but similar to that of thegroup treated with the F4-negative strain for most of the followingperiods, except days 14 to 18 (Table 12).

Other F4⁺ strains: The FI of all other F4⁺ strains was lower than thatof both control groups (groups 1 and 2) during the period days 4 to 9,early after the treatment (Table 12). Subsequently, the FI varied,depending on the strain (Table 12).

F4-negative strain: The FI of the group treated with an F4-negativestrain (group 2) was similar to that observed for the untreated group(group 1) until day 9, and was subsequently lower than the latter. Thus,the reduction of the FI for group 2 was observed later after treatmentthan for groups treated with F4⁺ strains.

The linear model showed a significant effect of the treatment on feedintake (p<0.0001). Post-hoc Dunnett's test showed that the feedconsumption was significantly higher for the control group (Group 1)than for the groups 2, 3, 4, and 7, treated with the 862, JFF4, JG1329,and pmK005 strains, respectively (Table 12). For these groups, mice ateless feed to reach the same weight.

3.5 Feed Conversion Ratio (FCR)

TABLE 13 Feed intake of mice after treatment with F4⁺ Escherichia coilstrains

^(*1)Treatment: Group 1; control, Group 2; F4-negative Escherichia colistrain at days 1 and 4, Groups 3 to 7; F4⁺ strains at days 1 and 4.

Although the difference was not significant, the FCR of the grouptreated with JFF4 was lower than that of any other treatment group fordays 0 to 14. Subsequently, the FCR was more variable between groups.The linear model showed no significant effect of the treatment on theFCR.

4. Analysis and Conclusion

The effect of F4⁺ Escherichia coli strains on growth seems to be lessimportant than that observed for pigs and chickens. Nevertheless, aneffect on the FI and the FCR was observed, particularly during thegrowth phase of the mice (first 14 days). Since CD-1 mice were notgenetically selected for growth performance, as was the case for thechickens and pigs used in the present studies, it is possible that themice rapidly reached the maximum weight gain and that there was thuslittle scope for the F4⁺ strains to influence weight gain, thusaffecting only feed intake.

Bernardeau et al. reported that the growth promoting effect ofLactobacilli was not observed in mice overfed with conventional enricheddiets whereas the impact of probiotic administration was enhanced inmice fed a sub-standard diet (such as one based on barley).

REFERENCES

-   Arnold S., Gassner B., Giger T. and Zwahlen R. Banning antimicrobial    growth promoters in feedstuffs does not result in increased    therapeutic use of antibiotics in medicated feed in pig farming.    Pharmacoepidemiology and Drug Safety 2004; 13: 323-331.-   Wegener H C, Aarestrup F M, Bogo Jensen L, Hammerum A M and Bager F.    Use of antimicrobial growth promoters in food animals and    Enterococcus faecium resistance to therapeutic antimicrobial drugs    in Europe. Emerging Infectious Diseases 1999; 5 (3): 329- 335.-   Schwarz S, Kehrenberg C and Walsh T R. Use of antimicrobial agents    in veterinary medicine and food animal production. International    Journal of Antimicrobial Agents 2001; 17: 431-437.-   Bernardeau M, Vernoux J P and Gueguen M. Safety and efficacy of    probiotic lactobacilli in promoting growth in post-weaning Swiss    mice. International Journal of Food Microbiology 2002; 77(1-2):    19-27.

1. Method of promoting growth of an animal, comprising the step of:feeding the animal with an F4+ non-pathogenic Escherichia coli strain,wherein the F4+ non-pathogenic Escherichia coli strain is in an amounteffective to promote growth of the animal.
 2. The method according toclaim 1, wherein the step of feeding comprises feeding orally.
 3. Themethod according to claim 2, wherein said F4+ non-pathogenic Escherichiacoli strain is in association with an acceptable carrier selected fromthe group consisting of a solid feed acceptable carrier, a liquid feedacceptable carrier, and a combination thereof.
 4. The method accordingto claim 3, wherein the solid feed acceptable carrier comprises a commonsolid feedstuff.
 5. The method according to claim 3, wherein the liquidfeed acceptable carrier comprises water.
 6. The method according toclaim 3, wherein the liquid feed acceptable carrier comprises milk. 7.The method according to claim 1, wherein the effective amount is atleast about 5×10⁷ CFU of F4+ non-pathogenic Escherichia coli strain peranimal.
 8. The method according to claim 1, wherein the effective amountranges from about 5×10⁷ to about 5×10⁹ CFU of F4+ non-pathogenicEscherichia coli strain per animal.
 9. The method according to claim 1,wherein the F4+ non-pathogenic Escherichia coli strain consists of theEscherichia coli strain deposited at the International DepositoryAuthority of Canada IDAC on Jan. 21, 2005 under accession number IDAC210105-01, mutants or variants thereof.
 10. The method according toclaim 1, wherein the animal is selected from the group consisting of apost-weaning animal, a post-hatching animal, and a combination thereof.11. The method according to claim 10, wherein the animal is apost-weaning animal aged from about 10 to about 28 days old.
 12. Themethod according to claim 10, wherein the animal is a post-hatchinganimal aged from about 1 to about 7 days old.
 13. The method accordingto claim 10, wherein the animal is a post-weaning pig.
 14. The methodaccording to claim 10, wherein the animal is a post-weaning mouse. 15.The method according to claim 10, wherein the animal is a post-hatchingpoultry animal.
 16. The method according to claim 15, wherein thepoultry animal is a chicken.
 17. The method according to claim 1,wherein said F4+ non-pathogenic Escherichia coli strain promotes dailyweight gain.
 18. The method according to claim 1, wherein said F4+non-pathogenic Escherichia coli strain promotes weight gain whilereducing feed intake.
 19. The method according to claim 1, wherein saidF4+ non-pathogenic Escherichia coli strain is fed to the animal duringthe animal's growth phase and reduces the delay required for the animalto attain a desired weight.